Immunity to human cytomegalovirus (HCMV) following infection is complex, and not fully protective against reactivation and reinfection with new strains. Although generally asymptomatic in immunocompetent adults, infection or reinfection in pregnant women can be devastating to the developing fetus if transplacental transmission occurs. Because of the lifelong disabilities caused by congenital CMV infection, such as sensorineural hearing loss (SNHL), understanding the host defense determinants that protect the developing fetus is critical, toward the goal of developing an effective preconception vaccine. Clinical trials of an adjuvanted glycoprotein B (gB) vaccine showed protection in young women of childbearing age, but waning immunity and modest efficacy (~50%) necessitate consideration of other subunit strategies. Recent evidence suggests that the pentameric complex (PC) of CMV proteins (gH/gL/UL128/UL130/UL131) may be a more compelling vaccine target for induction of protective antibody responses than gB. This stems from the observations that antibodies to the PC potently block virus entry into epithelial and endothelial cells and leukocytes. Moreover, acquisition of these antibodies correlates with a reduced risk of fetal transmission in women with primary CMV infection. To address whether a PC-based vaccine provides superior protection against congenital CMV transmission to that conferred by a gB-based vaccine, we will compare MVA- vectored gB and PC vaccines in the guinea pig model of congenital CMV infection, using the guinea pig CMV (GPCMV) homologs of these proteins. In addition to comparing the endpoints of maternal and pup mortality, congenital GPCMV infection, and magnitude of viral load following high-dose GPCMV challenge during pregnancy (aim 1), we will compare these vaccines for their ability to confer protection against SNHL (aim 2) following low-dose challenge during pregnancy. This study will represent the first animal model evaluation of a prenatal vaccine to prevent congenital CMV-induced labyrinthitis and SNHL. We will also address a second area of significant complexity in CMV vaccines, namely, addressing the phenomena of re-infection during pregnancy. It has become increasingly clear that, in spite of preconception immunity, women can become re-infected with new strains of HCMV, and these strains can be transmitted to the fetus, leading to injury. Therefore, in aim 3, we will utilze a newly discovered strain of GPCMV, the CIDMTR strain, to model re- infection studies in the guinea pig. We will test whether MVA-PC vaccination can provide superior protection compared to gB vaccine against re-infection and subsequent fetal transmission in dams with preconception immunity to a heterotypic strain, the ATCC (22122) strain. Since most congenital CMV infections occur in the context of non-primary maternal infections, these studies will substantially advance the field, and clarify what is required of a CMV vaccine in women of childbearing age.